Christopher S. Awtrey

Gene Expression Profiles Associated with Response to Chemotherapy in Epithelial Ovarian Cancers

Purpose: The goal of this study was to determine whether distinct gene expression profiles are associated with intrinsic and/or acquired chemoresistance in epithelial ovarian carcinoma. Experimental Design: Gene expression profiles were generated from 21 primary chemosensitive tumors and 24 primary chemoresistant tumors using cDNA-based microarrays. Gene expression profiles of both groups of primary tumors were then compared with those of 15 ovarian carcinomas obtained following platinum-based chemotherapy (“postchemotherapy” tumors). A theme discovery tool was used to identify functional categories of genes involved in drug resistance. Results: Comparison of primary chemosensitive and chemoresistant tumors revealed differential expression of 85 genes (P < 0.001). Comparison of gene expression profiles of primary chemosensitive tumors and postchemotherapy tumors revealed more robust differences with 760 genes differentiating the two groups (P < 0.001). In contrast, only 230 genes were differentially expressed between primary chemoresistant and postchemotherapy groups (P < 0.001). Common to both gene lists were 178 genes representing transcripts differentially expressed between postchemotherapy tumors and all primary tumors irrespective of intrinsic chemosensitivity. The gene expression profile of postchemotherapy tumors compared with that of primary tumors revealed statistically significant overrepresentation of genes encoding extracellular matrix–related proteins. Conclusions: These data show that gene expression profiling can discriminate primary chemoresistant from primary chemosensitive ovarian cancers. Gene expression profiles were also identified that correlate with states of intrinsic and acquired chemoresistance and that represent targets for future investigation and potential therapeutic interventions.

Microfilament-dependent activation of Na+/K+/2Cl- cotransport by cAMP in intestinal epithelial monolayers.

cAMP-mediated stimulation of Cl- secretion in the human intestinal cell line T84 is accompanied by significant remodeling of F-actin, and both the secretory and cytoskeletal responses may be largely ablated by previous cell loading with phalloidin derivatives, reagents that prevent dynamic reordering of microfilaments (1991. J. Clin. Invest. 87:1903-1909). In this study, we examined the effect of phalloidin loading on the cAMP-elicited activity of the individual membrane-associated transport proteins involved in electrogenic Cl- secretion. Efflux of 125I and 86Rb was used to assay forskolin-stimulated Cl- and K+ conductances, respectively, and no inhibitory effect of phalloidin could be detected. Na+/K(+)-ATPase pump activity, assessed as bumetanide-insensitive 86Rb uptake and the ability of monolayers to generate a Na+ absorptive current in response to apical addition of a Na+ ionophore, was not different between control and phalloidin-loaded monolayers. Forskolin was found to stimulate Na+/K+/2Cl- cotransport (bumetanide-sensitive 86Rb uptake) in time-dependent fashion. In the absence of any agonist, cotransporter activity was markedly decreased in phalloidin-loaded monolayers. Furthermore, under phalloidin-loaded conditions, the forskolin-elicited increase in bumetanide-sensitive 86Rb uptake was markedly attenuated. These findings suggest that cAMP-induced activity of Cl- channels, K+ channels, and the Na+/K(+)-ATPase are not influenced by F-actin stabilization. However, cAMP-induced activation of the Na+/K+/2Cl- cotransporter appears to be microfilament-dependent, and ablation of this event is likely to account for the inhibition of cAMP-elicited Cl- secretion seen in the phalloidin-loaded state. Such findings suggest that Na+/K+/2Cl- cotransporter is functionally linked to the cytoskeleton and is a regulated site of cAMP-elicited electrogenic Cl- secretion.

Current treatment for ovarian cancer.

Ovarian cancer is the most lethal gynaecologic malignancy. Epithelial ovarian cancer (EOC) constitutes approximately 90% of cases of ovarian cancer and 70% of the patients with EOC present in advanced stage. Treatment of EOC usually consists of cytoreductive surgery which includes total abdominal hysterectomy (TAH), bilateral salpingo-oophorectomy (BSO), omentectomy and lymphadenectomy followed by adjuvant chemotherapy. Current adjuvant chemotherapy includes paclitaxel and either cisplatin or carboplatin given every 3 weeks for six cycles. The combination paclitaxel and platinum chemotherapy achieves clinical response in approximately 80% of patients. However, most patients will have tumour recurrence within 3 years following treatment. Patients with platinum-sensitive tumours can be re-treated with platinum and/or paclitaxel. Those with platinum-resistant tumours have poor prognosis and treatment is palliative. Options of treatment in these patients include topotecan, doxil, gemcitabine, etoposide, or enrolment in clinical trials. Future research needs to focus on the role of cytoreductive surgery, second-look surgery, consolidation chemotherapy, development of new chemotherapeutic agents, chemoresistance modulators, as well as new approaches to the treatment of women with ovarian cancer.

Ammonia inhibits cAMP-regulated intestinal Cl- transport. Asymmetric effects of apical and basolateral exposure and implications for epithelial barrier function.

The colon, unlike most organs, is normally exposed to high concentrations of ammonia, a weak base which exerts profound and diverse biological effects on mammalian cells. The impact of ammonia on intestinal cell function is largely unknown despite its concentration of 4-70 mM in the colonic lumen. The human intestinal epithelial cell line T84 was used to model electrogenic Cl- secretion, the transport event which hydrates mucosal surfaces and accounts for secretory diarrhea. Transepithelial transport and isotopic flux analysis indicated that physiologically-relevant concentrations of ammonia (as NH4Cl) markedly inhibit cyclic nucleotide-regulated Cl- secretion but not the response to the Ca2+ agonist carbachol. Inhibition by ammonia was 25-fold more potent with basolateral compared to apical exposure. Ion substitution indicated that the effect of NH4Cl was not due to altered cation composition or membrane potential. The site of action of ammonia is distal to cAMP generation and is not due simply to cytoplasmic alkalization. The results support a novel role for ammonia as an inhibitory modulator of intestinal epithelial Cl- secretion. Secretory responsiveness may be dampened in pathological conditions associated with increased mucosal permeability due to enhanced access of lumenal ammonia to the basolateral epithelial compartment.

Assessment of long-term knowledge retention following single-day simulation training for uncommon but critical obstetrical events

Objective: The objectives were to determine (i) whether simulation training results in short-term and long-term improvement in the management of uncommon but critical obstetrical events and (ii) to determine whether there was additional benefit from annual exposure to the workshop. Methods: Physicians completed a pretest to measure knowledge and confidence in the management of eclampsia, shoulder dystocia, postpartum hemorrhage and vacuum-assisted vaginal delivery. They then attended a simulation workshop and immediately completed a posttest. Residents completed the same posttests 4 and 12 months later, and attending physicians completed the posttest at 12 months. Physicians participated in the same simulation workshop 1 year later and then completed a final posttest. Scores were compared using paired t-tests. Results: Physicians demonstrated improved knowledge and comfort immediately after simulation. Residents maintained this improvement at 1 year. Attending physicians remained more comfortable managing these scenarios up to 1 year later; however, knowledge retention diminished with time. Repeating the simulation after 1 year brought additional improvement to physicians. Conclusion: Simulation training can result in short-term and contribute to long-term improvement in objective measures of knowledge and comfort level in managing uncommon but critical obstetrical events. Repeat exposure to simulation training after 1 year can yield additional benefits.

Description and Validation of the Pelv-Sim: A Training Model Designed to Improve Gynecologic Minimally Invasive Suturing Skills

STUDY OBJECTIVE To describe and validate the Pelv-Sim trainer, an innovative training model for gynecologic laparoscopic suturing with 4 laparoscopic exercises: closing an open vaginal cuff, transposing an ovary to the pelvic sidewall, ligating an infundibulopelvic ligament, and closing a port-site fascial incision. DESIGN Randomized controlled trial (Canadian Task Force classification I). SETTING Academic medical center. PARTICIPANTS Obstetrics and gynecology residents (n = 19) and third-year medical students (n = 10). INTERVENTIONS To test the Pelv-Sim model for construct validity, all participants were timed as they completed the 4 tasks, and their performances were compared. The residents were then randomized to a study group asked to train with the Pelv-Sim for 1 hour/week for 10 weeks, or to a control group. To evaluate the effectiveness of training with the Pelv-Sim model, both groups of residents were retested at the end of the 10-week study period. Pretraining and posttraining performances were compared within each group. MEASUREMENTS AND MAIN RESULTS Before the intervention, the residents completed all 4 tasks in significantly less time than the medical students (all p values <or=.012). When retested after the 10-week study period, the control group showed no significant performance improvements. The trained group showed significant improvement in performance for the vaginal cuff closure task (p = .004) and the ovary transposition task (p = .047), but not for the infundibulopelvic ligament ligation or the fascial closure tasks. CONCLUSION Construct validity was shown for all 4 Pelv-Sim simulation tasks. Resident training improves performance on the vaginal cuff closure and ovary transposition tasks. The Pelv-Sim has the potential to be a valuable tool in laparoscopic training for gynecology residents.

Urinary incontinence, depression, and economic outcomes in a cohort of women between the ages of 54 and 65 years.

OBJECTIVE: To estimate the association between urinary incontinence (UI) and probable depression, work disability, and workforce exit. METHODS: The analytic sample consisted of 4,511 women enrolled in the population-based Health and Retirement Study cohort. The analysis baseline was 1996, the year that questions about UI were added to the survey instrument, and at which time study participants were 54–65 years of age. Women were followed-up with biennial interviews until 2010–2011. Outcomes of interest were onset of probable depression, work disability, and workforce exit. Urinary incontinence was specified in different ways based on questions about experience and frequency of urine loss. We fit Cox proportional hazards regression models to the data, adjusting the estimates for baseline sociodemographic and health status variables previously found to confound the association between UI and the outcomes of interest. RESULTS: At baseline, 727 participants (survey-weighted prevalence, 16.6%; 95% confidence interval [CI] 15.4–18.0) reported any UI, of which 212 (survey-weighted prevalence, 29.2%; 95% CI 25.4–33.3) reported urine loss on more than 15 days in the past month; and 1,052 participants were categorized as having probable depression (survey-weighted prevalence, 21.6%; 95% CI 19.8–23.6). Urinary incontinence was associated with increased risks for probable depression (adjusted hazard ratio, 1.43; 95% CI 1.27–1.62) and work disability (adjusted hazard ratio, 1.21; 95% CI 1.01–1.45), but not workforce exit (adjusted hazard ratio, 1.06; 95% CI 0.93–1.21). CONCLUSION: In a population-based cohort of women between ages 54 and 65 years, UI was associated with increased risks for probable depression and work disability. Improved diagnosis and management of UI may yield significant economic and psychosocial benefits. LEVEL OF EVIDENCE: II

Dynamic role of microfilaments in intestinal chloride secretion

The importance of microfilaments in the regulation of chloride (Cl-) secretion by the human intestinal cell line T84 was investigated using the cytoskeletal probe phalloidin to bind and stabilize F-actin. Phalloidin was found to inhibit secretion mediated by cyclic adenosine monophosphate (cAMP) and the sustained secretory response to the calcium (Ca+2) ionophore ionomycin but not to affect the transient Ca+2-mediated response to carbachol and histamine. Fluorescent microscopic examination of F-actin revealed regionally restricted microfilament remodeling in cAMP- and ionomycin-treated cells. Normal regulation of apical Cl- and basolateral potassium (K+) channel functions was evident in phalloidin-loaded cells. It is concluded that prevention of cytoskeletal remodeling by actin stabilization inhibits the generation of a sustained Cl- secretory response by a mechanism that does not involve Cl- or K+ channels. Depolymerization of F-actin plays an integral role in the regulation of intestinal Cl- secretion.

Clinically inapparent invasive vulvar carcinoma in an area of persistent Paget’s disease: a case report

BACKGROUND Invasive vulvar carcinoma is reported to occur in 5 to 20% of patients with vulvar Pagets disease. We report a case in which a clinically inapparent invasive lesion was discovered on reexcision of microscopically persistent vulvar Pagets disease. CASE A 58-year-old woman presented with a diagnosis of vulvar Pagets disease. A wide local excision of the lesion was performed and pathologic analysis revealed microscopic Pagets disease at two of the margins. The patient returned for a follow-up 4 months later and a vulvar biopsy revealing persistent Pagets cells was obtained from the area of the prior microscopically positive surgical margin. A reexcision was performed from the normal-appearing vulva and invasive vulvar carcinoma was noted in this specimen. CONCLUSIONS This case demonstrates several concerning aspects of this disease, most important of which is that the clinically apparent lesion did not contain the clinically significant invasive lesion. Invasive vulvar carcinoma may occur in association with microscopically persistent vulvar Pagets disease, a condition often encountered after primary treatment with wide local excision.

Safety and Outcome of Patients Treated with a Modified Outpatient Intraperitoneal Regimen for Epithelial Ovarian, Primary Peritoneal or Fallopian Tube Cancer

Background: Despite the survival benefit of intraperitoneal (IP) chemotherapy observed in GOG172, significant toxicity and poor treatment completion rates have prevented the widespread acceptance of this regimen. Here, we report our experience with a modified outpatient GOG172 regimen. Methods: Eligible patients had stage III, optimally debulked epithelial ovarian, fallopian tube or primary peritoneal cancer that underwent IP port placement for administration of a modified GOG172 regimen consisting of: (i) intravenous paclitaxel 135 mg/m2 on day 1 over 3 h; (ii) intraperitoneal cisplatin 75 mg/m2 on day 2, and (iii) intraperitoneal paclitaxel 60 mg/m2 on day 8. Day 8 IP paclitaxel was omitted until tolerance of the first cycle of IP cisplatin had been established. Results: Four or more cycles of IP chemotherapy were completed by 72.5% (29) of 40 eligible patients; 20% of patients exhibited catheter-related complications requiring port removal and discontinuation of IP chemotherapy. Grade 3-4 hematologic, metabolic and gastrointestinal toxicities occurred in 36, 8 and 21% of the patients, respectively. With a median follow-up of 47.7 months, progression-free and overall survival was comparable to GOG172. Conclusions: This modified outpatient GOG172 regimen is associated with less toxicity and improved completion rates compared to the original GOG172 regimen.